4. MATERIALS AND METHODS
6.2 Effects of antioxidative plant phenolics on oxidation of tryptophan (III, IV)
6.2.1 Tryptophan oxidation in the presence of oilseed byproducts
Camelina, rapeseed and soy meal phenolics revealed a more pronounced effect in inhibiting the hexanal/FeCl2 induced tryptophan oxidation (IV) than H2O2 induced oxidation of tryptophan (III). In general, since tryptophan was less oxidized in the presence of camelina meal, rapeseed meal, and soy phenolics in both studies (III, IV), the formation of tryptophan oxidation products was delayed, which consequently resulted in more pronounced inhibitions toward primary and secondary oxidation products. In contrast, pine bark phenolics were more effective antioxidants in H2O2 oxidized (III) tryptophan than when oxidized with hexanal/FeCl2 (IV). Therefore, also the ability of pine bark phenolics in hexanal/FeCl2 model to inhibit the formation of tryptophan derived oxidation products was weakened.
The camelina meal phenolics comprise of flavonols, hydroxycinnamic acids and flavanols, which presumably have a synergistic effect. For example, in study IV the antioxidant activity of the principal reference compounds in camelina meal such as sinapic acid and catechin toward loss of tryptophan was weaker than that of the camelina meal extract. In addition, quercetin and chlorogenic acid showed either no effect or prooxidant activity toward tryptophan loss. However, in study III, it was shown that individual reference compounds such as catechin, quercetin, sinapic acid and chlorogenic acid at different concentration levels were able to inhibit the oxidation of tryptophan with similar or even better activities than the extracts themselves. According to study II, quercetin contributed to the antioxidant effect of camelina meal. As rapeseed meal contained sinapine as the main component, it may also be the effective form contributing to antioxidant activity. This would be in accordance with studies I and II, where rapeseed phenolics inhibited both protein and lipid oxidation in cooked pork meat patties. Rapeseed phenolics have been reported to show moderate radical scavenging activity (DPPH test) and inhibit oxidation of liposomes (Vuorela et al., 2005a).
Flavanols and isoflavones are the most important phenolic compounds present in soy. The effect of individual isoflavones such as genistein and daidzein on tryptophan oxidation was more pronounced in H2O2 oxidized tryptophan than in hexanal/FeCl2 model. This data shows
that the antioxidant activity of individual phenolic compounds and oilseed byproducts toward oxidation tryptophan is dependent on the oxidant present.
The comparison between the total phenolic content measured by Folin-Ciocalteau method and the total amount of specific phenolic groups or phenolic compounds measured by HPLC methods showed that there are differences between the methods (Table 6). It is known that Folin-Ciocalteau method reacts strongly with all reducing hydroxyl groups present not only in phenolic compounds but also in some proteins and sugars (Singleton and Rossi, 1965).
These other highly reactive compounds include tertiary aliphatic amines, primary, secondary, and tertiary aromatic amines, tryptophan, hydroxylamine, hydrazine, certain purines, and other miscellaneous organic and inorganic reducing agents (Ikawa, 2003). Therefore, this procedure usually leads to an overestimation of the total polyphenolic content. Therefore, the separation and identification carried out by HPLC methods gives a more specific data on phenolic groups and phenolic compounds. The results also showed that there were variations between the phenolic compositions among similar extracts used in different studies (Table 6). This is due to that the materials used in different studies originated from different batches.
Therefore, the variations in composition (phenolics, protein, lipids) are most likely due to environmental factors such as light, temperature and humidity as well as genetics of the plant.
It can be concluded that the results of phenolic profiles obtained by HPLC methods give a more accurate and reliable data on the phenolic compositions compared to total phenolic content obtained by Folin-Ciocalteau procedure. In addition, the processing (extraction) methods have a great impact on the phenolic composition as have been described in other studies (Matthaus, 2002; Vuorela et al., 2003; 2004).
Pine bark phenolics consist mainly of flavanols (~80 µg/g) with catechin dominating. In study IV, catechin at 100 µM was able to inhibit tryptophan loss by 10%, whereas taxifolin, another phenolic compound reported in pine bark, showed either no effect or weak prooxidant activity. However, in study III when tryptophan was oxidized by H2O2, catechin and taxifolin protected tryptophan from oxidation. Flavonols (e.g. quercetin) and flavanones (e.g. taxifolin) have been shown to have higher metal-initiated prooxidant activity (Cao et al., 1997). This may explain why reference compounds quercetin and taxifolin as such exhibited prooxidant or no effect toward loss of tryptophan when iron was added to the model (IV).
Based on this data, pine bark phenolics showed either antioxidant or prooxidant effects toward tryptophan oxidation depending on the oxidant.
In conclusion, for the first time camelina, rapeseed and soy meal as well as pine bark rich in diverse phenolics were shown to be potential antioxidant towards oxidation of amino acid tryptophan. However, as the results showed, this is highly dependent on the oxidant used in the model. As the data showed, the pure phenolic compounds used as reference compounds did not explicate the exact activity of certain plant extract. It can be hypothized that the network of phytochemicals is essential for the activity of plant materials, particularly when considering that a plant antioxidant may become a pro-oxidant if suitable and sufficient co-antioxidants are missing. Most of the active co-antioxidants are likely to be pro-oxidants when they lie beyond the optimum. In addition, the antioxidant activity of certain compound(s) contributing to activity is difficult to demonstrate since there may also be synergistic effects between the different phenolics present in the extracts.
6.2.2 Tryptophan oxidation in the presence of berry phenolics
Black currant anthocyanins showed the best protection toward tryptophan loss when oxidized with H2O2 (III). In contrast, when oxidized with hexanal/FeCl2, black currant was not able to inhibit the oxidation of tryptophan. In addition, raspberry anthocyanins were not effective in either model (III, IV). Rowanberry phenolics were not able to inhibit the oxidation of tryptophan (IV). Berry phenolics that were not able to protect tryptophan from oxidation yielded also more oxidation products regardless of the oxidant used. Black currant contains four major anthocyanins: the 3-glucosides and 3-rutinosides of cyanidin (7 and 38%) and delphinidin (16 and 39%), whereas raspberry consists mainly of the sophorisides (59%), 3-glucosides (16%), and 3-glucosylrutinosides (16%) of cyanidin with minor amounts of pelargonidin (4%) with different 3-glucosyl substituents (Viljanen et al., 2005b). Cyanidin-3-glucoside and delphinidin-3-Cyanidin-3-glucoside were able to inhibit the tryptophan loss by 30% (III).
Effects of cyanidin-3-glucoside and delphinidin-3-glucoside (although with less effect) showed a consistency in the pattern of oxidation products formed with black currant and raspberry anthocyanins (III). Therefore, according to these results, it seems that cyanidin- and delphinidin-3-glucosides present are the main compounds responsible for the antioxidant activities in black currant isolates. In study IV, however, cyanidin-3-glucoside showed prooxidant activity toward tryptophan loss.
It has been reported that black currant consists also of procyanidins (43% of total proanthocyanidins), and prodelphinidins (57%) with low molecular weight (LMW) (1-10
µg/g) and insoluble high molecular weight (HMW) (100 µg/g) proanthocyanidins, which may also contribute to the antioxidant activity (Ferreira et al., 2006). Prodelphinidins in the form of trimeric gallocatechins (Ferreira et al., 2006) and flavan-3-ols such as catechin (8 µg/g) and epicatechin (11 µg/g) have been identified from black currant (Määtta-Riihinen et al., 2004a). This suggests that the amount of 3.7% of flavanols in black currant isolate may also affect the oxidation of tryptophan, which is in accordance with the results that catechin as a reference compound inhibited the tryptophan oxidation (III, IV).
It has been shown that 3-glucosides and 3-rutinosides of cyanidin and dephinidin can act as lipid and protein antioxidants in liposomes and oil-in-water emulsions (Viljanen et al., 2004a;
2005b). In addition, black currant and raspberry anthocyanin isolates have been shown to inhibit protein and lipid oxidation in liposomes (Viljanen et al., 2004b). In another study, black currant anthocyanins were reported to be better antioxidants toward protein and lipid oxidation in oil-in-water emulsions than raspberry anthocyanins (Viljanen et al., 2005b). The antioxidant properties of flavonoids are mainly due to the 3′, 4′-dihydroxy group located on the B ring, the 3-hydroxy or 5-hydroxy and the 4-carbonyl groups in the C-ring (Fernandez et al., 2002). In addition, the antioxidant activity increases with the number of hydroxyl groups in rings A and B. The inability to protect tryptophan from oxidation may be due to that anthocyanins have a very low oxidation potential (spontaneous oxidation) which renders them into either pro-oxidants by redox-cycling, or good antioxidants depending on the reaction conditions (Van Acker et al., 1996).
Raspberry ellagitannins showed a weak antioxidant activity toward tryptophan oxidation when oxidized with H2O2 (III), but a prooxidant activity when oxidized with hexanal/FeCl2
(IV). The main compounds in ellagitannin fraction consist of mixture of monomers (MW 936 g/mol), dimers (sanguiin H6), trimers (lambertianin C), and polymers (Kähkönen et al., unpublished results). Raspberry is reported to contain minor amounts of flavonols such as 3-glucosides and 3-glucuronides of quercetin (Määtta-Riihinen et al., 2004b), which was in accordance with our results (III, IV). Ellagic acid and raspberry ellagitannins have been attributed with antioxidative properties (Vuorela et al., 2005a). Ellagic acid with increasing concentration exhibited the best activity against oxidation of tryptophan (III) by decreasing the tryptophan loss by 50%. Ellagic acid, however, was not able to inhibit tryptophan loss when oxidized with hexanal/FeCl2 (IV). It is known that the affinities of tannins for binding,
crosslinking and consequently precipitating proteins are dependent on the structural flexibility of both the tannin and protein molecule (Deaville et al., 2007). The loss of conformational freedom of ellagitannins significantly affects their binding capability (Deaville et al., 2007). In addition, it has been shown that metal ions catalyze the oxidation and polymerization of the phenolic compounds therefore reducing their available binding-sites (Dangles et al., 2006). It may be concluded that the effects of raspberry ellagitannins and ellagic acid toward oxidation of tryptophan depends on the oxidant used. The presence of hexanal/FeCl2 in the tryptophan solution renders both ellagitannins and ellagic acid to act as prooxidants. Ellagic acid, however, acted as antioxidant when H2O2 is present. In addition, based on this data as well as knowledge on literature it may be that the ability of ellagic acid as superior antioxidant compared to ellagitannins may be due to their structural differences, or perhaps synergistic properties with other compounds.
Functions of cranberry proanthocyanidins were investigated only when tryptophan was oxidized by H2O2 (III). In this model cranberry proanthocyanidins were among the best phenolics that inhibited the oxidation of tryptophan. Cranberry procyanidin fractions have been found to be effective antioxidants when using DPPH test and toward lipid oxidation inhibiting the oxidation of methyl linoleate emulsion and LDL (Määttä-Riihinen et al., 2005).
Cranberry, blueberry, and grape seed extracts alone and in combinations showed antioxidant activity assayed by using a DPPH radical inhibition test (Vattem et al., 2005a). In addition, cranberry juice powder and its synergies with ellagic and rosmarinic acids have been shown to reduce oxidative stress and mediate antioxidant enzyme responses in porcine muscle tissue induced by H2O2 oxidation (Vattem et al., 2005b). This data suggests that cranberry proanthocyanidins are effective in inhibiting the oxidation of tryptophan. In addition, according to literature the antioxidant activity of cranberries have also been proven in other oxidation models. Therefore, cranberries could be used in many different food applications to improve the oxidative stability.
In conclusion, based on the data obtained combinations of antioxidants i.e. extracts of berry and oilseed phenolics are more effective in preventing oxidative degradation in tryptophan than single compounds. However, the effectiveness is dependent on the oxidation model used as was also concluded before. The concentration ratios of different phenolic compounds in the plant extracts may be critical for their activity. This may explain the differences in efficacy for camelina and rapeseed meals compared to rowanberry phenolics even though
they comprised of similar phenolics. In addition, it may be that the concentration levels of the phenolics used in studies III and IV were not optimal for the antioxidant activity, especially when the concentrations of the phenolics (10, 50 or 100 µM) were very low compared to the tryptophan concentration (2 mM). More systematic research is needed to optimize the levels of phenolics to be the most effective and to further explicate their antioxidant effect toward protein oxidation by identifying the unknown compounds formed during the oxidation.
The indole i.e. pyrrole moiety of tryptophan is the most likely group to be involved in the reaction with the phenolic compounds since the oxidation of the indole structure yielding N-formylkynurenine and kynurenine was effectively inhibited by oilseed byproducts in studies III and IV, and by berry phenolics in study III. This is in accordance with a study (Rawel et al., 2001) proposing that the semiquinones or quinones of phenolic compounds may react with the heterocyclic nitrogen-atom of tryptophan. The antioxidant activity of plant phenolics may be due to the ability of flavonoid semiquinones or quinones in binding directly to tryptophan, and thereby preventing it from further reactions. Further oxidation of this product can lead to formation of tryptophan dimers or longer polymers. Another possibility is the reaction between flavonoid radical and tryptophyl radical. However, flavonoid termination reactions do not necessary lead to termination of radical scavenging since oxidation products (dimers or quinones) and their degradation products may still be reactive (Seyoum et al., 2006). Based on the results, it may be concluded that the most important target for antioxidant action of plant phenolics was preventing the cleavage of the indole moiety of tryptophan. Consequently, the overall oxidation of tryptophan was then inhibited. The exact antioxidant mechanism, however, remains unclear, and needs to be investigated in the future studies.